The long term goal of this project is to understand the molecular mechanisms of alcohol's actions. Due to its small size and lack of chemical features, alcohol is often considered to be a relatively non-specific drug. Many cellular factors have been identified that contribute to regulating cellular responses to ethanol that ultimately lead to the development of addiction. However, it is clear that alcohol does not interact with all these proteins, but in fact shows a relatively high degree of specificity for a limited number of receptors, ion channels and other signaling molecules. This led to the hypothesis that in many cases alcohol acts by binding to specific sites in these proteins to bring about changes in protein structure and function. However, there is very little knowledge about what makes up an alcohol binding site and contribute to alcohol sensitivity. Previously, we identified a specific alcohol binding site in the Drosophila protein LUSH. Structural and biochemical studies of this protein led to the discovery that concerted interactions from multiple hydrogen bonding resides were required to form an ethanol binding site. In this work we will focus on determining the interactions between alcohol and regulatory domains of Protein Kinase C (PKC) family of enzymes. Several isoforms of PKC proteins play a central in regulating responses to ethanol. Further, several isoforms of PKC have been shown to be directly sensitive to alcohol and it is suggested that ethanol can modulate binding of important cofactors required for sub-cellular localization and activation. We will use structural biology methods to determine how alcohol interacts with these domains, and how these interactions lead to changes in structure and dynamics that could alter the ability of PKC domains to interact with co-factors. We have already collected preliminary data that have identified a specific interaction between alcohol and PKC-alpha. Finally we propose to investigate the interactions between alcohol and the transmembrane domains of the glycine receptor. Glycine receptors are one of the primary targets of alcohol in the central nervous systems, and a wealth of studies have developed a model for how alcohol may affect the function of this receptor. Our studies will determine what residues are critical for binding alcohol, an how the known mutations are likely to its modify alcohol sensitivity PUBLIC HEALTH RELEVANCE Understanding how ethanol acts to modulate protein structure and function is a fundamental problem relevant to human health. Defining the sites of alcohol actions in proteins, and how amino acids in these sites contribute to ethanol sensitivity, will provide a greater understanding of the mechanism of alcohol's actions. This may allow future development of novel approaches to prevent alcohols actions, and the identification of genetic markers associated with a predisposition to alcoholism.